Isolation of transcripts over-expressed in human pathogen Trichophyton rubrum during growth in keratin.

Trichophyton rubrum is a cosmopolitan and anthropophilic fungus able to invade keratinized tissue, causing infection in human skin and nails. This work evaluated the changes in the extracellular pH during its growth in keratin (after 6, 12, 24, 48, 72h and 7 days) at initial pH 5.0. We observed a gradual increase of basal pH under keratin exposure when compared to glucose condition. Also, we identified 576T. rubrum transcripts differentially expressed by subtractive suppression hybridization (SSH) using conidia cultivated for 72h in keratin as tester, and cultivated in glucose as driver. The over-expression of 238 transcripts obtained under keratin condition was confirmed by macro-array dot-blot, revealing 28 unigenes. Putative proteins encoded by these genes showed similarity to fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, NIMA interactive protein, Gag-Pol polyprotein, virulence factors serine-protease subtilisin and metalloprotease, cytochrome P450, GlcN-6-phosphate deaminase and Hsp30. The upregulation of T. rubrum genes encoding subtilisin, metalloprotease and Gag-Pol polyprotein was also validated by northern blot. The results of this study provide the first insight into genes differentially expressed during T. rubrum grown in keratin that may be involved in fungal pathogenesis.